Pretherapeutic Expression of the hOCT1 Gene Predicts a Complete Molecular Response to Imatinib Mesylate in Chronic-Phase Chronic Myeloid Leukemia

In this retrospective study we evaluated the pretherapeutic mRNA expression of the hOCT1 (human organic cation transporter 1) gene in patients with chronic-phase (CP) chronic myeloid leukemia (CML) who varied in terms of their response to imatinib (IM). hOCT1 mRNA was quantified by real-time PCR. Patients were classified as expressing either high (n = 44) or low hOCT1 mRNA (n = 44). The complete cytogenetic response rates observed at 6, 12 and 18 months were 47.7, 84.1 and 91%, respectively, in patients with high hOCT1 mRNA and 47.5, 81.8 and 86.3%, respectively, in patients with low hOCT1 transcripts. The major molecular response rates were not significantly different between patients with high and low hOCT1 mRNA after 6 months of therapy (22.7 vs. 9.1%; p = 0.07), but they were significantly different after 12 months (54.5 vs. 31.8%; p = 0.026) and 18 months (77.2 vs. 56.8%; p = 0.034). Complete molecular responses were observed in 5 patients with low and 17 patients with high hOCT1 mRNA (p = 0.003). The 5-year event-free and overall survival analyses revealed no significant differences between the groups. These data imply that knowledge of the pretherapeutic level of hOCT1 could be a useful marker to predict IM therapy outcome in treatment-naive CP CML patients. Copyright (C) 2012 S. Karger AG, Basel

CYP3A5 and NAT2 gene polymorphisms: role in childhood acute lymphoblastic leukemia risk and treatment outcome

Susceptibility to acute lymphoblastic leukemia can be highly influenced by genetic polymorphisms in metabolizing enzyme genes of environmental carcinogens. This study aimed to evaluate the impact of the CYP3A5 and NAT2 metabolizing enzyme polymorphisms on the risk of childhood acute lymphoblastic leukemia. The analysis was conducted on 204 ALL patients and in 364 controls from a Brazilian population, using PCR-RFLP. The CYP3A5*3 polymorphic homozygous genotype was more frequent among ALL patients and the *3 allele variant was significantly associated with increased risk of childhood ALL (OR = 0.29; 95% CI, 0.14-0.60). The homozygous polymorphic genotype for the *6 allele variant was extremely rare and found in only two individuals. The heterozygous frequencies were similar for the ALL group and the control group. No significant differences were observed between the groups analyzed regarding NAT2 variant polymorphisms. None of the polymorphisms analyzed was related to treatment outcome. The results suggest that CYP3A5*3 polymorphism may play an important role in the risk of childhood ALL.

Molecular basis for the diagnosis and treatment of acute promyelocytic leukemia

Acute promyelocytic leukemia is characterized by gene rearrangements that always involve the retinoic acid receptor alpha on chromosome 15. In the majority of patients t(15;17) is detected, which generates the promyelocytic leukemia gene/retinoic acid receptor alpha rearrangement. This rearrangement interacts with several proteins, including the native promyelocytic leukemia gene, thus causing its delocalization from the nuclear bodies, impairing its function. The immunofluorescence staining technique using the anti-PML antibody may be used to provide a rapid diagnosis and to immediately start therapy using all-trans retinoic acid. The experience of the International Consortium on Acute Promyelocytic Leukemia has demonstrated that early mortality was significantly reduced by adopting the immunofluorescence technique. All-trans retinoic acid combined with chemotherapy is the standard therapy; this promotes complete remission rates greater than 90% and cure rates of nearly 80%. However, early mortality is still an important limitation and hematologists must be aware of the importance of treating newly diagnosed acute promyelocytic leukemia as a medical emergency.

A novel myeloid leukemia suppressor locus at human chromosome 5q13.3

Molecular mechanisms that underlie preleukemic myelodysplasia (MDS) and acute myelogenous leukemia (AML) are poorly understood. In MDS or AML with a refractory clinical course, more than 30% of patients have acquired interstitial or complete deletions of chromosome 5. The 5q13.3 chromosomal segment is commonly lost as the result of 5q deletion. Reciprocal and unbalanced translocations of 5q13.3 can also occur as sole anomalies associated with refractory AML or MDS. This study addresses the hypothesis that a critical gene at 5q13.3 functions either as a classical tumor suppressor or as a chromosomal translocation partner and contributes to leukemogenesis. ^ Previous studies from our laboratory delineated a critical region of loss to a 2.5–3.0Mb interval at 5q13.3 between microsatellite markers D5S672 and GATA-P18104. The critical region of loss was later resolved to an interval of approximately 2Mb between the markers D5S672 and D5S2029. I, then generated a long range physical map of yeast artificial chromosomes (YACs) and developed novel sequence tagged sites (STS). To enhance the resolution of this map, bacterial artificial chromosomes (BACs) were used to construct a triply linked contig across a 1 Mb interval. These BACs were used as probes for fluorescent in situ hybridization (FISH) on an AML cell line to define the 5q13.3 critical region. A 200kb BAC, 484a9, spans the translocation breakpoint in this cell line. A novel gene, SSDP2 (single stranded DNA binding protein), is disrupted at the breakpoint because its first four exons are encoded within 140kb of BAC 484a9. This finding suggests that SSDP2 is the critical gene at 5q13.3. ^ In addition, I made an observation that deletions of chromosome 5q13 co-segregate with loss of the chromosome 17p. In some cases the deletions result from unbalanced translocations between 5q13 and 17p13. It was confirmed that the TP53 gene is deleted in patients with 17p loss, and the remaining allele harbors somatic mutation. Thus, the genetic basis for the aggressive clinical course in AML and MDS may be caused by functional cooperation between deletion or disruption of the 5q13.3 critical gene and inactivation of TP53. ^

Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults

Reduction of 5,10-methylenetetrahydrofolate (methyleneTHF), a donor for methylating dUMP to dTMP in DNA synthesis, to 5-methyltetrahydrofolate (methylTHF), the primary methyl donor for methionine synthesis, is catalyzed by 5,10-methylenetetrahydrofolate reductase (MTHFR). A common 677 C → T polymorphism in the MTHFR gene results in thermolability and reduced MTHFR activity that decreases the pool of methylTHF and increases the pool of methyleneTHF. Recently, another polymorphism in MTHFR (1298 A → C) has been identified that also results in diminished enzyme activity. We tested whether carriers of these variant alleles are protected from adult acute leukemia. We analyzed DNA from a case–control study in the United Kingdom of 308 adult acute leukemia patients and 491 age- and sex-matched controls. MTHFR variant alleles were determined by a PCR-restriction fragment length polymorphism assay. The MTHFR 677TT genotype was lower among 71 acute lymphocytic leukemia (ALL) cases compared with 114 controls, conferring a 4.3-fold decrease in risk of ALL [odds ratio (OR = 0.23; 95% CI = 0.06–0.81]. We observed a 3-fold reduction in risk of ALL in individuals with the MTHFR 1298AC polymorphism (OR = 0.33; 95% CI = 0.15–0.73) and a 14-fold decreased risk of ALL in those with the MTHFR 1298CC variant allele (OR = 0.07; 95% CI = 0.00–1.77). In acute myeloid leukemia, no significant difference in MTHFR 677 and 1298 genotype frequencies was observed between 237 cases and 377 controls. Individuals with the MTHFR 677TT, 1298AC, and 1298CC genotypes have a decreased risk of adult ALL, but not acute myeloid leukemia, which suggests that folate inadequacy may play a key role in the development of ALL.

A bcr-3 isoform of RARα-PML potentiates the development of PML-RARα-driven acute promyelocytic leukemia

Acute promyelocytic leukemia (APML) most often is associated with the balanced reciprocal translocation t(15;17) (q22;q11.2) and the expression of both the PML-RARα and RARα-PML fusion cDNAs that are formed by this translocation. In this report, we investigated the biological role of a bcr-3 isoform of RARα-PML for the development of APML in a transgenic mouse model. Expression of RARα-PML alone in the early myeloid cells of transgenic mice did not alter myeloid development or cause APML, but its expression significantly increased the penetrance of APML in mice expressing a bcr-1 isoform of PML-RARα (15% of animals developed APML with PML-RARα alone vs. 57% with both transgenes, P < 0.001). The latency of APML development was not altered substantially by the expression of RARα-PML, suggesting that it does not behave as a classical “second hit” for development of the disease. Leukemias that arose from doubly transgenic mice were less mature than those from PML-RARα transgenic mice, but they both responded to all-trans retinoic acid in vitro. These findings suggest that PML-RARα drives the development of APML and defines its basic phenotype, whereas RARα-PML potentiates this phenotype via mechanisms that are not yet understood.

Human AML1/MDS1/EVI1 fusion protein induces an acute myelogenous leukemia (AML) in mice: A model for human AML

The human t(3;21)(q26;q22) translocation is found as a secondary mutation in some cases of chronic myelogenous leukemia during the blast phase and in therapy-related myelodysplasia and acute myelogenous leukemia. One result of this translocation is a fusion between the AML1, MDS1, and EVI1 genes, which encodes a transcription factor of approximately 200 kDa. The role of the AML1/MDS1/EVI1 (AME) fusion gene in leukemogenesis is largely unknown. In this study, we analyzed the effect of the AME fusion gene in vivo by expressing it in mouse bone marrow cells via retroviral transduction. We found that mice transplanted with AME-transduced bone marrow cells suffered from an acute myelogenous leukemia (AML) 5–13 mo after transplantation. The disease could be readily transferred into secondary recipients with a much shorter latency. Morphological analysis of peripheral blood and bone marrow smears demonstrated the presence of myeloid blast cells and differentiated but immature cells of both myelocytic and monocytic lineages. Cytochemical and flow cytometric analysis confirmed that these mice had a disease similar to the human acute myelomonocytic leukemia. This murine model for AME-induced AML will help dissect the molecular mechanism of AML and the molecular biology of the AML1, MDS1, and EVI1 genes.

Acquired, nonrandom chromosomal abnormalities associated with the development of acute promyelocytic leukemia in transgenic mice

We previously generated a transgenic mouse model for acute promyelocytic leukemia (APL) by expressing the promyelocytic leukemia (PML)–retinoic acid receptor (RARα) cDNA in early myeloid cells. This fusion protein causes a myeloproliferative disease in 100% of animals, but only 15–20% of the animals develop acute leukemia after a long latency period (6–13 months). PML-RARα is therefore necessary, but not sufficient, for APL development. The coexpression of a reciprocal form of the fusion, RARα-PML, increased the likelihood of APL development (55–60%), but did not shorten latency. Together, these results suggested that additional genetic events are required for the development of APL. We therefore evaluated the splenic tumor cells from 18 transgenic mice with APL for evidence of secondary genetic events, by using spectral karyotyping analysis. Interstitial or terminal deletions of the distal region of one copy of chromosome 2 [del(2)] were found in 1/5 tumors expressing PML-RARα, but in 11/13 tumors expressing both PML-RARα and RARα-PML (P < 0.05). Leukemic cells that contained a deletion on chromosome 2 often contained additional chromosomal gains (especially of 15), chromosomal losses (especially of 11 or X/Y), or were tetraploid (P ≤ 0.001). These changes did not commonly occur in nontransgenic littermates, nor in aged transgenic mice that did not develop APL. These results suggest that expression of RARα-PML increases the likelihood of chromosome 2 deletions in APL cells. Deletion 2 appears to predispose APL cells to further chromosomal instability, which may lead to the acquisition of additional changes that provide an advantage to the transformed cells.

The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-MLL/FBP17-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

RIG-E, a human homolog of the murine Ly-6 family, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell.

In vivo all-trans-retinoic acid (ATRA), a differentiation inducer, is capable of causing clinical remission in about 90% of patients with acute promyelocytic leukemia (APL). The molecular basis for the differentiation of APL cells after treatment with ATRA remains obscure and may involve genes other than the known retinoid nuclear transcription factors. We report here the ATRA-induced gene expression in a cell line (NB4) derived from a patient with APL. By differential display-PCR, we isolated and characterized a novel gene (RIG-E) whose expression is up-regulated by ATRA. The gene is 4.0 kb long, consisting of four exons and three introns, and is localized on human chromosome region 8q24. The deduced amino acid sequence predicts a cell surface protein containing 20 amino acids at the N-terminal end corresponding to a signal peptide and an extracellular sequence containing 111 amino acids. The RIG-E coded protein shares some homology with CD59 and with a number of growth factor receptors. It shares high sequence homology with the murine LY-6 multigene family, whose members are small cysteine-rich proteins differentially expressed in several hematopoietic cell lines and appear to function in signal transduction. It seems that so far RIG-E is the closest human homolog of the LY-6 family. Expression of RIG-E is not restricted to myeloid differentiation, because it is also present in thymocytes and in a number of other tissues at different levels.

Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia.

Chromosomal rearrangements involving band 12p13 are found in a wide variety of human leukemias but are particularly common in childhood acute lymphoblastic leukemia. The genes involved in these rearrangements, however, have not been identified. We now report the cloning of a t(12;21) translocation breakpoint involving 12p13 and 21q22 in two cases of childhood pre-B acute lymphoblastic leukemia, in which t(12;21) rearrangements were not initially apparent. The consequence of the translocation is fusion of the helix-loop-helix domain of TEL, an ETS-like putative transcription factor, to the DNA-binding and transactivation domains of the transcription factor AML1. These data show that TEL, previously shown to be fused to the platelet-derived growth factor receptor beta in chronic myelomonocytic leukemia, can be implicated in the pathogenesis of leukemia through its fusion to either a receptor tyrosine kinase or a transcription factor. The TEL-AML1 fusion also indicates that translocations affecting the AML1 gene can be associated with lymphoid, as well as myeloid, malignancy.

Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns.

Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD(+) acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD(+) human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD(+) allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD(+) patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.

Tyrosine kinase inhibitor insensitivity of non-cycling CD34+ human acute myeloid leukaemia cells with FMS-like tyrosine kinase 3 mutations.

The efficacy of tyrosine kinase (TK) inhibitors on non-cycling acute myeloid leukaemia (AML) cells, previously shown to have potent tumourigenic potential, is unknown. This pilot study describes the first attempt to characterize non-cycling cells from a small series of human FMS-like tyrosine kinase 3 (FLT3) mutation positive samples. CD34+ AML cells from patients with FLT3 mutation positive AML were cultured on murine stroma. In expansion cultures, non-cycling cells were found to retain CD34+ expression in contrast to dividing cells. Leukaemic gene rearrangements could be detected in non-cycling cells, indicating their leukaemic origin. Significantly, the FLT3-internal tandem duplication (ITD) mutation was found in the non-cycling fraction of four out of five cases. Exposure to the FLT3-directed inhibitor TKI258 clearly inhibited the growth of AML CD34+ cells in short-term cultures and colony-forming unit assays. Crucially, non-cycling cells were not eradicated, with the exception of one case, which exhibited exquisite sensitivity to the compound. Moreover, in longer-term cultures, TKI258-treated non-cycling cells showed no growth impairment compared to treatment-naive non-cycling cells. These findings suggest that non-cycling cells in AML may constitute a disease reservoir that is resistant to TK inhibition. Further studies with a larger sample size and other inhibitors are warranted.

Synthetic lethal targeting of oncogenic transcription factors in acute leukemia by PARP inhibitors

Acute myeloid leukemia (AML) is mostly driven by oncogenic transcription factors, which have been classically viewed as intractable targets using small molecule inhibitor approaches. Here, we demonstrate that AML driven by repressive transcription factors including AML1-ETO and PML-RARα are extremely sensitive to Poly (ADP-ribose) Polymerase (PARP) inhibitor (PARPi), in part due to their suppressed expression of key homologous recombination genes and thus compromised DNA damage response (DDR). In contrast, leukemia driven by MLL fusions with dominant transactivation ability is proficient in DDR and insensitive to PARP inhibition. Intriguing, depletion of an MLL downstream target, Hoxa9 that activates expression of various HR genes, impairs DDR and sensitizes MLL leukemia to PARPi. Conversely, Hoxa9 over-expression confers PARPi resistance to AML1-ETO and PML-RARα transformed cells. Together, these studies describe a potential utility of PARPi-induced synthetic lethality for leukemia treatment and reveal a novel molecular mechanism governing PARPi sensitivity in AML.